Capillary Gas Chromatography-selected Ion Monitoring Research Articles

A GC-SIM-MS Method for the Determination of Butylidenephthalide in Rat Plasma and Tissue: Application to the Pharmacokinetic and Tissue Distribution Study

Butylidenephthalide is one of the major active components isolated from Rhizoma Chuanxiong. This paper describes a simple, rapid, specific and sensitive method for the quantification of butylidenephthalide in rat plasma and tissue distribution using a liquid-liquid extraction procedure followed by capillary gas chromatography-selected ion monitoring mode-mass spectrometry (GC-SIM-MS) analysis. The calibration curves were linear over the concentration ranging from 0.02–10.0 µg/mL (r > 0.99) for plasma samples and 0.18–7.25 µg/g (r > 0.99) for the tissue samples. The limit of quantification (LOQ) was 1.0 ng/mL or 1.0 ng/g (ten times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.39–2.98% and 2.97–4.26%, respectively. The methods of recovery for all samples were greater than 80% at the low, medium, and high concentrations. The method has been successfully applied to a pharmacokinetics study in rats after an oral administration of Butylidenephthalide with a dose of 20.0 mg/kg. The main pharmacokinetic parameters obtained were T max = (0.22 ± 0.06) h, C max = (3 ± 1) µg/mL, AUC = (32 ± 6) h•µg/mL, and K a = (8.5 ± 0.8)/h. The results showed that the butylidenephthalide was easily absorbed. The concentrations of butylidenephthalide in rat kidney, lung, heart, and cerebellum were higher than those in other organs. To determine free fraction in serum, samples were filtered using ultrafiltration membranes with a molecular weight cut-off of 10,000 Da and extracted using liquid-liquid extraction. The extracts were evaporated and analyzed by GC-MS. The protein binding in rat plasma, human plasma, and human serum albumin were 83 ± 4%, 94 ± 3%, and 89 ± 3%, respectively.

A capillary gas chromatography-selected ion monitoring mass spectrometry method for the analysis of atractylenolide I in rat plasma and tissues, and application in a pharmacokinetic study

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC–MS method. All bio-samples were cleared up with a liquid–liquid extraction procedure. The calibration curves were linear within a range of 5–1000 ng/mL for plasma samples, 0.06–16.00 μg/g for cerebellum samples, and 0.03–8.00 μg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N ≥ 10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: T max = 0.37 ± 0.19 h, C max = 0.26 ± 0.05 μg/mL, AUC = 1.95 ± 0.30 μg h/mL and k a = 10.08 ± 5.60 h −1. The tissue distribution of AO-I in rats after the oral administration of 50.0 mg/kg was from 0.225 to 0.031 μg/g with a decreasing tendency in different tissues like liver > kidney > spleen > cerebellum > heart > cerebrum > lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8 ± 3.9, 90.6 ± 3.1 and 60.9 ± 5.1%, respectively.

Evaluation of D-amino acid levels in rat by gas chromatography-selected ion monitoring mass spectrometry: no evidence for subacute toxicity of orally fed D-proline and D-aspartic acid.

Sprague-Dawley rats received deionized water (controls) during 28 days or drinking water with added D-proline, L-proline, D-aspartic acid or L-aspartic acid corresponding to a mean daily load of approximately 50 mg amino acid enantiomer kg-1 body weight. Parameters indicating the physiological status (food intake and body weight, glutamic-oxalic-transaminase, glutamic-pyruvic-transaminase, alkaline phosphatase, urea and creatinine in serum, and creatine and osmomolality of urine) were determined. After 28 days the weights of the supposed target organs of toxicity (kidney, liver, brain, thymus) were determined and organs were inspected for macroscopic and microscopic alterations. No pathological changes in the organs were observed and no signs of subacute toxicity (liver, kidney) were found. In serum, homogenates of liver, kidney and brain, and in part, in urine, the amounts of D-amino acids (D-AAs) were quantitatively determined using chiral phase capillary gas chromatography-selected ion monitoring mass spectrometry. Significant levels of certain D-AAs (Ala, Pro, Ser, Asx, Glx, Orn and Lys) were already detectable in kidney and liver homogenates and serum of controls. In brain homogenates the highest amounts among the D-AAs were found for D-Ser (up to 382 nmol g-1), moderate amounts for D-Ala, D-Asx and D-Glx, and, in a few cases, trace amounts for D-Orn and D-Lys (1-2 nmol g-1). D-Pro was not detected either in the brains of controls or in the brains of animals loaded with D-Pro. Feeding with D-Pro resulted in a 20-30 fold increased renal excretion of D-Pro at the end of the experiment. Continuous feeding with D-Asp did not increase renal excretion of this enantiomer, but in the serum, higher amounts (0.8-4.0 mumol-1) were determined in comparison to the control group (0.3-0.9 mumol-1). Feeding with D-Pro led to an increase of this enantiomer in serum (1.3-10.5 mumol-1). Feeding with D-Asp did not increase its amounts in brain homogenates (38 and 43 nmol g-1) in comparison to controls.

Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 Seedlings of Zea mays L.

Gibberellins A(12) (GA(12)), GA(53), GA(44), GA(19), GA(17), GA(20), GA(29), GA(1), and GA(8) have been identified from extracts of vegetative shoots of normal (wild type) maize using full scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Seven of these gibberellins (GAs) have been quantified by capillary gas chromatography-selected ion monitoring using internal standards of [(14)C(4)]GA(53), [(14)C(4)]GA(44), [(2)H(2)] GA(19), [(13)C(1)]GA(20), [(13)C(1)]GA(29), [(13)C(1)]GA(1), and [(13)C(1)]GA(8). Quantitative data from extracts of normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings support the operation of the early 13-hydroxylation pathway in vegetative shoots of Zea mays. These data support the positions in the pathway blocked by the mutants, previously assigned by bioassay data and metabolic studies. The GA levels in dwarf-2, dwarf-3, and dwarf-5 were equal to, or less than, 2.0 nanograms per 100 grams fresh weight, showing that these mutants are blocked for steps early in the pathway. In dwarf-1, the level of GA(1) was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA(20) and GA(29) accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA(20) to GA(1). Since the level of GAs beyond the blocked step for each mutant is greater than zero, each mutated gene probably codes for an altered gene product, thus leading to impaired enzyme activities.

Identification of gibberellins A 1 and A 19 from Populus balsamifera x P. deltoides

Gibberellins (GAs) A 1 and A 19 were identified from young shoots of a naturally occurring interspecific poplar hybrid ( Populus balsamifera x P. deltoides). Detection of GA-like substances was accomplished by the Tanginbozu dwarf rice microdrop assay after sequential SiO 2 partition chromatography and reversed-phase C 18 HPLC with internal standards of [ 1H]GA 1 and [ 3H]GA 20. The radioactive and/or bioactive peaks were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM) with internal standards of [17, 17- 2H]GAs A 1, and A 19. Identifications were thus based on retention times relative to authentic standards on three sequential chromatographic systems as well as the relative intensifies of characteristic ions on GC-SIM. Similar analysis by bioassay and GC-SIM with [17, 17- 2H]GA 20 also suggested the presenceof GA 20.

Gibberellins and gravitropism in maize shoots: endogenous gibberellin-like substances and movement and metabolism of [3H]Gibberellin A20.

[3H]Gibberellin A20 (GA20) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [3H]GA20 to [3H]GA1- and [3H]GA29-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [3H]GA1, [3H]GA29, and [3H]GA8. The tentative identification of these putative [3H]GA glucosyl conjugates was further supported by the release of the free [3H]GA moiety after cleavage with cellulase. Within 12 hours of the [3H]GA20 feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [3H]GA20, especially [3H]GA1, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the "Tanginbozu" dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA20-like substance and lower halves containing principally a GA20-like substance and lower halves containing mainly GA1-like and GA19-like substances. Gibberellins A1 (10 nanograms per gram) and A20 (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.

Metabolism of [3H]Gibberellin A5 by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A(5) (GA(5)) as 1- and 1,2-[(3)H]GA(5) (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [(3)H]GA-like metabolites were separated from the highly H(2)O-soluble [(3)H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted --> isocratic eluted reversed-phase C(18) high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [(3)H] GA(5) were identified as GA(1), GA(3), and GA(6) by GC-SIM. The major highly water soluble metabolite of [(3)H]GA(5) at all levels of substrate GA(5) had chromatographic characteristics similar to authentic GA(1)-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [(3)H]GA(5) feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA(1), GA(3), GA(5) methyl ester, GA(6), GA(22), GA(29) (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA(1), GA(3), GA(5), and GA(8) (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA(6) and GA(29) would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA(5). Increasing substrate GA(5) levels increased the proportion of metabolites with HPLC Rts similar to GA(1), GA(6), and especially GA(1) glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA(3), GA(3)-glucosyl ester, and a postulated conjugate of GA(6). There was evidence that high doses of substrate GA(5) induced new metabolites which often, but not always, differed from GA(1), GA(3), and GA(6) in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M(+) and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.

Comparison of the endogenous gibberellins in the shoots and roots of vernalized and non-vernalized chinese spring wheat seedlings

Endogenous gibberellins (GAs) in Chinese Spring wheat seedlings were isolated by high performance liquid chromatography (HPLC) and identified by combined capillary gas chromatography-selected ion monitoring (GC-SIM). Gibberellins A 1, A 3, A 19, A 20, A 44, and A 53 were identified in the shoots, A 19 and A 20 in the roots. The identification of these 13-hydroxylated GAs demonstrates the presence of the early-13-hydroxylation pathway in wheat seedlings. Based on peak area of total ion response of five characteristic ions by GC-SIM, the approximate levels of GAs in the shoots is GA 44 > GA 19 > GA 1 = GA 3 > GA 20 for the non-vernalized wheat seedlings, and GA 44 > GA 19 > GA 53 = GA 3 > GA 1 = GA 20 for the vernalized wheat seedlings. The C 20 GAs, GA 53, GA 44 and GA 19, are present in shoots of the vernalized (flowering) wheat seedlings at somewhat higher levels than that in the non-vernalized (rapidly growing) wheat seedlings. Approximate levels of the C 19 GAs, GA 20, GA 1 and GA 3 were lower in the shoots of the vernalized wheat seedlings than in the non-vernalized wheat seedlings. The conversion of GA 19 to GA 20 (C 20 to C 19 GAs) may be a rate-limiting step in the vernalized wheat seedlings.

Metabolism of [ 3H]gibberellin A 5 in developing pharbitis nil seeds

The native gibberellin A 5 (GA 5), as [1- 3H]GA 5 (3.2 Ci/mmol) was fed to seed capsules (0.58 μCi/capsule) of Pharbitis nil cv Violet at the 2-week stage of development, and its metabolism in the seeds was investigated after 43 hr. Extractable radioactivity in free GA metabolites was 38%, with 56% in GA glucosyl conjugate-like substances. Only 2.5% of the extractable radioactivity remained as [ 3H]GA 5. Tentative identifications, based on comparisons with authentic standards after sequential chromatography on silica gel partition column → gradient-eluted C 18 HPLC → isocratic-eluted C 18 HPLC-radiocounting (RC), showed that [ 3H]GA 5 was converted to at least six free GAs, GA 1, GA 3, GA 6, GA 8, GA 22, GA 29, a GA 5 methyl ester-like metabolite, and at least twelve GA glucosyl conjugate-like substances, GA 5-glucoside (GA 5-G), GA 5-glucosyl ester (GA 5-GE), GA 1-O(3)-G, GA 1-O(13)-G, GA 1-GE, GA 3-O(3)-G, GA 3-O(13)-G, GA 3-GE, GA 6-G or GE, GA 8-O(2)-G, GA 22-G or GE and GA 29-O(2)-G. After lower specific activity feeds of [1,2- 3H]GA 5 (74 mCi/mmol; 0.1 μCi/capsule) at approximately the same stage of development, the presence of GA 1, GA 3, GA 5, GA 6, GA 8 and GA 29 was further confirmed by sequential (after C 18 HPLC-RC) capillary gas chromatography-selected ion monitoring (GC-SIM), using six characteristic ions. However, for GA 22 only a trace of the parent ion was present at the appropriate retention time.

Gas Chromatography-Selected Ion MonitoringによるFM-100投与後の血中グリチルリチン及びグリチルレチン酸の定量

Glycyrrhizin and glycyrrhetinic acid in the human plasma after administration of FM-100 (the powder obtained from acidic fraction of methanolic extract of Glycyrrhiza) were determined by capillary gas chromatography-selected ion monitoring by using [2H5]glycyrrhetinic acid methyl ester as an internal standard. Plasma was subjected to direct hydrolysis, and the resulting mixture was adsorbed on a polyamide column. Glycyrrhetinic acid was eluted with methanol, and the eluant was further purified by a Sep-Pak Silica cartridge and a silicagel column after the addition of the internal standard. Glycyrrhetinic acid, a metabolite of glycyrrhizin, was directly extracted with AcOEt from the plasma with high recovery. Glycyrrhetinic acid extracted was converted to its methyl ester-O-trimethylsilyl ether derivative by treating with diazomethane and then with N, O-bis-(trimethylsilyl) trifluoroacetamide. Monitoring of the molecular ion indicated that the quantitation limit for glycyrrhizin was 1 ng/ml plasma. After oral administration of 400 mg of FM-100, plasma levels of glycyrrhizin and glycyrrhetinic acid were shown with a maximum at 8-14 h post dose period. For the study of bioavailability of glycyrrhizin, each subject was indispensable to fast in order to avoid the effect of natural glycyrrhizin from many food additives for more than 10 h before and after administration of FM-100, respectively. This method was useful for the study on pharmacokinetics of glycyrrhizin and glycyrrhetinic acid, because of having sufficient sensitivity and specificity for objective compounds.

Microanalysis of bile acid in human liver tissue by selected ion monitoring

A method of microquantitative determination of bile acid in 5–30 mg of human liver tissue was developed. Bile acids were converted to their ethyl ester dimethylethylsilyl ether derivatives and analyzed by capillary gas chromatography-selected ion monitoring, using [ 2H 4]-lithocholic (LCA), [ 2H 5]deoxycholic (DCA), [ 2H 4]chenodeoxycholic (CDCA), [ 2H 4]-ursodeoxycholic (UDCA), and [ 2H 3]cholic (CA) acids as internal standards. Precision and reproducibility of the present method were tested using surgically obtained liver specimens. The results were statistically analyzed according to one-way layout and the orthogonal polynomial equation. Bile acids except LCA were determined with 2.3 to 11.4% of the coefficient of variation. Recoveries of conjugated bile acids ranged from 72.2 to 96.0% with a mean of 84.3%. The amount of bile acids present in histologically normal liver specimens ( n = 10) was found to be 29.56 ± 8.62 μg/g liver. The relative compositions (%) of CDCA and CA were 38.8 ± 8.9 and 41.1 ± 11.0, respectively.